Endotoxin adsorbent for the prevention and treatment of autoimmune diseases

ABSTRACT

It is believed that the abnormal absorption of endotoxin present in the gastrointestinal tract relates to the pathogenesis of autoimmune diseases such as rheumatoid arthritis. In an animal model for rheumatoid arthritis, it is observed that arthritis is improved by removing endotoxin. The present invention provides an endotoxin-adsorbent, which is capable of removing endotoxin in the gastrointestinal tract and can be administered to humans safely. By using a non-digestible and non-absorbable, and therefore, safe endotoxin-adsorbent, which has a high endotoxin-binding capacity for removing large amounts of endotoxin present in the gastrointestinal tract, it is possible to prevent and treat autoimmune diseases such as rheumatoid arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior U.S. application Ser. No.11/518,393, filed Sep. 7, 2006 now U.S. Pat. No. 7,799,347.

FIELD OF THE INVENTION

This invention relates to an endotoxin-adsorbent for preventing andtreating autoimmune diseases, such as rheumatoid arthritis, by removingendotoxin in the gastrointestinal tract.

RELATED ARTS

Lipopolysaccharide (LPS), a component of the outer cell membrane ofgram-negative bacteria, is known as endotoxin; and the lipid A componentis the fatal toxic domain of LPS (Microbiology. David B D, Dulbecco R,Eisen E N, Harold S, Ginsberg H S, Barry W A. Harper InternationalEdition 615-617, 1970).

Endotoxin has a variety of physiological and pathological effects andcauses endotoxin shock in animals within one hour if enough amounts ofLPS were injected. Since it causes fever even at low dose, it is alsoknown as a pyrogen. Therefore, the contamination of endotoxin in medicalproducts such as injection products used as a non-oral administration isstrictly prevented.

Since large numbers of a variety of gram-negative microorganisms, suchas E. coli, invariably reside in the gastrointestinal tract of animalsand humans, a large amount of endotoxin consistently is present in thegastrointestinal tract. However, animals and humans do not suffer afever in general, indicating that endotoxin is barely absorbed fromintestinal walls due to its large molecular size or due to mucosalimmune barrier systems such as the IgA antibody barrier on intestinalwalls.

The immune system is one of the major self-defense systems for a host tokeep homeostasis by recognizing and preventing the invasion of foreignsubstances such as microorganisms as well as the growth of abnormalcells such as cancer cells, and by excluding them from the body.However, once this system is destroyed for unknown reasons, the immunesystem starts to attack self-components, and as a consequence, induces avariety of intractable diseases, so called “autoimmune diseases”. Thefollowing diseases are known as autoimmune diseases: rheumatoidarthritis, autoimmune hepatitis, autoimmune nephritis, autoimmunelabyrinthitis, autoimmune encephalomyelitis, autoimmune chronicthyroiditis, type I diabetes, systemic lupus erythematosus,polydermatomyositis, psoriasis, Sjorgren syndrome, ulcerative colitis,Crohn's disease, and Guillain-Barre syndrome.

Rheumatoid arthritis is an example of a typical autoimmune disease.Since large numbers of patients suffer this painful disease and theirquality of life in society is interfered with by their limited function,this disease has been given a lot of social attention. The majority ofpatients with rheumatoid arthritis are forced to be confined to bed restas the progress of arthritis, such as articular destruction, jointdeformity, mobility impairment and pain increases. Currently, autoimmunediseases are treated with non-steroidal anti-inflammatory drugs,anti-inflammatory steroids, immune suppressants, and anti-cytokineantibodies such as Remicade. These therapeutic agents only suppressabnormal immune systems and inflammatory reactions, and are used totarget specific symptoms, but not intended to cure the disease. Althoughseveral hypotheses for the causes of rheumatoid arthritis have beenproposed, the etiology and the pathology of this disease remain unknown.

Based on the analysis of auto-antibodies in sera and cartilages frompatients with rheumatoid arthritis, we have reached a hypothesis thatthe chronic abnormal absorption of mimic antigens and bacterial toxinsfrom the gastrointestinal tract due to increased mucosal permeability isthe fundamental, common disorder of autoimmune diseases. This hypothesiswas proved by the following arthritis models in experimental animals. Weadministered purified heterologous type II collagen with and without theuse of LPS to mice by the oral route, and successfully induced threetypes of chronic arthritis in mice. Most importantly, bacterial toxins,such as endotoxin, are not only capable of disturbing immune homeostasisby stimulating host immune systems non-specifically, but also capable ofinducing inflammatory diseases such as arthritis. Based on theseobservations, the inventors of this invention focused on the pathogenicroles of LPS, which is a dominant bacterial toxin produced by intestinalflora in large quantities, and reached a hypothesis that autoimmunediseases could be prevented and treated by blocking the absorption ofexcess amounts of LPS from the gastrointestinal tract by using anendotoxin-adsorbent.

To begin, mice were administered chick type II collagen by the oralroute for more than 10 weeks. Mice developed antibodies to chick type IIcollagen, which cross-reacts to autologous type II collagen, and as aconsequence, mice developed clinically apparent arthritis (Terato K, YeX Y, Miyahara H, Cremer M A, and Grifiths M M. Induction of auto-immunearthritis in DBA/l mice by oral administration of type II collagen. Br.J. Rheum. 35:828-838, 1996).

Since autoantibodies to cartilage are not always capable of inducingarthritis in experimental animals and humans, it was assumed that asecondary factor(s) is involved in the induction of arthritis inpatients with rheumatoid arthritis. Although a variety of bacterialtoxins are considered as a potential secondary pathogenic factor, it ismost likely that endotoxin will play the dominant pathological role inthe majority of patients with autoimmune diseases, because endotoxin isthe most widely and commonly existing at high levels in thegastrointestinal tract. In order to test this possibility, mice wereinjected with a non-arthritogenic dose of monoclonal anti-type IIcollagen antibody cocktail and then received LPS by IP and oral route.The control mice receiving anti-type II collagen antibody alone did notdevelop arthritis, whereas, both groups of mice receiving LPS by IP andby oral administration developed severe arthritis, indicating thatenvironmental factors such as LPS play important pathological roles inautoimmune diseases (Terato K, Harper D S, Griffiths M M, Hasty I D A,Ye X Y, Cremer M A and Seyer J S. Collagen-induced arthritis:Synergistic effect of E. coli lipopolysaccharide bypass epitopespecificity in the induction of arthritis with monoclonal antibodies totype II collagen. Autoimmunity 22:137-147, 1995).

It has been known that endotoxin is not only involved in rheumatoidarthritis but also involved in a variety of autoimmune diseases such asautoimmune encephalomyelitis (Nagai A et al. J. Immunol. 175:959-966,2005), lupus lung injury (Chae B S et al. Arch Pharm Res 29:302-309,2006), autoimmune thyroiditis (Damotte I D et al. Fur Cytokine Netw14:52-59, 2003), primary biliary liver cirrhosis (Ballet E et al. J.Autoimmun 22:153-158, 2004), and Guillain-Barre syndrome (Yuki N et al.Proc Natl Acad Sci USA 101:11404-11409, 2004).

The inventors of this invention have shown previously that oraladministration of anti-LPS antibodies effectively suppressed thedevelopment of arthritis in this arthritis model (JP-A2006-151914). Thisevidence suggests strongly that the removal of endotoxin from thegastrointestinal tract is one of the best strategies for the treatmentof autoimmune diseases.

There are a variety of difficulties in mass-producing an antibody formedical use, in addition to the high production cost. Antibody, which isa protein, is heat labile, and tends to lose biological activity duringprocessing. Furthermore, an antibody administered by the oral route willbe less effective because of its degradation by digestion enzymes in thegastrointestinal tract. Therefore, it is desired to develop a new andeffective endotoxin antagonist which is heat-stable, easy tomass-produce at a low cost and safe for humans.

The contamination of endotoxin in injectable products must be removedcompletely, since even a minor contaminant of endotoxin induces adverseeffects such as fever in patients. In order to remove endotoxin frommedical products, several adsorbents specific to endotoxin have beenused. Synthetic fibers, fabrics and particles covalently bound by asubstance that has a high binding affinity to endotoxin, have been used.By contacting these adsorbents with an objective solution, endotoxincontaminated in the solution can be removed effectively. Severalendotoxin-adsorbents, such as Affi-Prep Polymixin (BioRad, USA) andToraymyxin (Toray Medicals, Japan), JP-A 11-335396 and JP-A 2002-263486,are currently used to remove endotoxin contaminated in injectableproducts and others.

SUMMARY OF THE INVENTION

The present invention provides a non-digestible and non-absorbableendotoxin-adsorbent, used for oral administration, comprising particlesin which not more than 1% have a diameter of not more than 5 μm, andmore than 90% of the particles have a diameter of not more than 50 μm,based on a volume-based size distribution analysis.

Furthermore, this invention also provides a formulation of theendotoxin-adsorbent or an agent for preventing and treating autoimmunediseases, a method for prevention and treatment of patients withautoimmune diseases by administering the endotoxin-adsorbent, andmethods for the manufacture of endotoxin-adsorbents used for theprevention and treatment of autoimmune diseases.

DETAILED DESCRIPTION OF THE INVENTION

Endotoxin-adsorbent used for the treatment of patients with autoimmunediseases requires the following criteria:

1) a high endotoxin-binding capacity, 2) suitable physiological featuresfor oral administration usage and 3) a high margin of safety without anyadverse effects. The number of microorganisms residing in the humangastrointestinal tract is believed to be approximately 100 trillion, andthe number of endotoxin-containing microorganisms among these bacteriais also massive. Therefore, in order to remove large portions ofendotoxin in the gastrointestinal tract, the endotoxin-adsorbent musthave a high binding capacity of endotoxin.

In order to satisfy these requirements, new endotoxinadsorbents, whichhave a high endotoxin-binding capacity, high margin of safety, andsuitable physical features for oral administration use, are provided inthis invention.

The toxic core of an endotoxin molecule is located in the Lipid Aregion. This invention consists of a Lipid A-binding substance andinsoluble carrier particles in order to adsorb and eliminate largeamounts of endotoxin from the gastrointestinal tract into feces by oraladministration, for preventing and treating autoimmune diseases such asrheumatoid arthritis.

The endotoxin-adsorbent suitable for the usage of above purposes is asfollows:

1. Endotoxin binding capacity of the particles is not less than 10×10⁶endotoxin units (EU) per 1 g of dry particles in an in-vitro test tubeassay.

2. Endotoxin binding capacity of the particles is not less than 50×10⁶endotoxin units (EU) per 1 g of dry particles in an in-vitro test tubeassay.

3. Endotoxin binding capacity of the particles is not less than 100×10⁶endotoxin units (EU) per 1 g of dry particles in an in-vitro test tubeassay.

4. The endotoxin-adsorbent consists of an endotoxin-binding substanceand carrier particles.

5. The endotoxin-adsorbent consists of a Lipid A binding substance, thatis capable of binding endotoxin.

6. The endotoxin-adsorbent, which is a Lipid A binding substance, ispolymixin B.

7. The endotoxin-adsorbent consists of a Lipid A binding substance,polymixin B, and carrier particles which are a weakly acidic cationexchange resin with a carboxy residue.

8. The methods for prevention and treatment of autoimmune diseases byadministrating pharmacologically effective doses of anendotoxin-adsorbent to patients by the oral route. Several materialssuch as polymixin B or a peptide antibiotic, and endotoxin-bindingpeptides (JP-A 11-335396, JP-A 2002-263486, JP-A 2002-311029, JP-A2004-292357 and JP-A 2002-512140) have been known as Lipid A-bindingsubstances. All these materials can be used for the purpose of thisinvention.

The carrier of the endotoxin-binding substance is desired to be smallparticles or powder suitable and convenient for oral administrationusage. A variety of polysaccharides and their derivatives, such ascellulose, agarose, mannan, glucan, and chitin, or a variety ofsynthetic polymers, such as polyacrylate, polystyrene, polypropylene,polyamide, and polyvinyl, can be used as the carrier of anendotoxin-binding substance.

There is no definite method required to conjugate the Lipid A bindingsubstance to the carrier particles. Cross-linkers which have been widelyused for immobilizing enzymes onto the solid surfaces, likewater-soluble carbodiimides such as ECDI, hexamethylene diisocyanate,propyleneglycol di-glycidylether, which contains 2 epoxy residues, andepichlorohydrin, can be used.

The features of Lipid A-binding substance-carrier complexes are desiredto be non-toxic, non-absorbable, and non-digestible by digestion enzymesand by microorganisms and resistant to other intestinal components suchas gastric acid. “Non-digestible” means resistant to both digestionenzymes of animals and enzymes produced by microorganisms.

The microorganisms residing in the gastrointestinal tract possessenzymes which are capable of digesting cellulose and other substancesthat are resistant to the digestion enzymes of animals (Kopecny J et al.Detection of cellulolytic bacteria from the human colon. Folia Microbiol(Praha) 49:175-7, 2004, Nakajima N et al. Dietary-fiber-degradingenzymes from a human intestinal Clostridium and their application tooligosaccharide production from nonstarchy polysaccharide usingimmobilized cells. Appl Microbiol Biotechnol 59: 182-9, 2002).Therefore, in this invention, the materials used as a carrier of theLPS-binding substance should be restricted to materials, which areresistant to bacterial digestion, and usage of polysaccharides such ascellulose, agarose, mannan, glucan, and chitin as a carrier of anendotoxin-binding substance should be excluded.

Compared to naturally occurring polymers, synthetic polymers aregenerally resistant to digestion enzymes secreted into thegastrointestinal tract of animals and even to various bacterial enzymes.Therefore, it is desired to choose a synthetic polymer as a carrier ofthe lipid A-binding substance. In fact, synthetic polymers such aspolystyrene sulfonate calcium and anion exchange resin are widely usedas a potassium adsorbent and as a cholesterol adsorbent for treatment ofpatients with high potassium and high cholesterols, respectively.

The particle size of the endotoxin-adsorbent is an important factor thatshould be considered, since it has been known that small size particles,such as yeast, of less than 5 μm in diameter, are phagocytized by Mcells, which reside on the surface of Peyer's patches scattered alongsmall and large bowel regions (Gerbert A. et al. M cells in Peyer'spatches of the intestine. Int Rev Cytol. 167:91-159, 1996). Therefore, aparticle size of endotoxin-adsorbent of not more than 5 μm in a diameteris excluded according to the specification of polystyrene sulfonatecalcium defined in Japanese Pharmacopoeia.

The molecular weight of endotoxin is more than 10,000 daltons, andassumed to bind mainly on the surfaces of endotoxin-adsorbent particlesrather than the inside of the particles. Therefore, if the particle sizeis smaller, the endotoxin-binding capacity is larger due to the largersurface area per unit weight of particles. This evidence is shown inEXAMPLE 12.

There are two classes of fine grinding techniques, dry and wet methods.The impact method, screen method, grind method and others are known asdry methods, whereas the catalyst-stirring method is an example of atypical wet method. There are several other methods, but there is nolimitation in the methods for grinding the particles of theendotoxin-adsorbent, and any of these methods can be used for preparingfine powder or small particles of the endotoxin-adsorbent.

The particle size of the endotoxin-adsorbent was determined based on theparticle size distribution method. The particle distribution analysiswas performed according to “The method for determining particle sizedistribution. Method 1: Microscopic method” in the second supplement ofthe general test procedures, section 65, Japanese Pharmacopoeia, 13^(th)Issue. The 50% particle size (μm) was explained as the diameter ofparticles of the corresponding values of the accumulative volume ofparticles is 50%.

The LPS binding capacity of the endotoxin-adsorbent in a test tube wasdetermined according to the method described in “endotoxin testprocedures” in Japanese Pharmacopoeia, in addition to a simple assaymethod of LPS by measuring OD values, which was developed during thisinvention. The experimental procedures and results are shown in EXAMPLE10 and 11 in detail.

The therapeutic effect of the endotoxin-adsorbent on autoimmune diseasescan be determined in the mouse arthritis model as described in ourprevious invention, JP-A 2006-151914. Briefly, arthritis can be inducedin 100% of mice by IP injection of enough amounts of anti-type IIcollagen monoclonal antibody cocktail (Chondrex Inc., Redmond, Wash.,USA) within 3 days (Terato K et al. Induction of arthritis withmonoclonal antibodies to collagen. J. Immunol. 148:2103-2108, 1992). Byreducing the dose of the monoclonal antibody cocktail to 2 mg, all miceremained normal without developing arthritis. However, oraladministration of 3 mg of LPS on 3 consecutive days from day 0, day 1,day 2 and day 3 into these mice induced clinically apparent arthritis,which reached the peak on day 6-7. Furthermore, one group of mice wasco-administered with indomethasin and ovoinhibitor, a protease-inhibitorpurified from egg white. The combination of indomethasin andovoinhibitor was used to increase the mucosal permeability ofgastro-intestinal mucosa. In these mice, the effect of LPS was moresignificant, and more severe arthritis was induced by oraladministration of a same dose of LPS. Using this arthritis model inducedby a combination of monoclonal antibody and LPS, the therapeutic effectof the endotoxin-adsorbent can be determined. The benefit of this modelis multifold: time of experimentation is short compared with anauthentic collagen-induced arthritis model, the standard deviation ofseverity of arthritis among individual mice is much less, and the effectof the LPS-adsorbent is clearly determined.

Since the endotoxin-adsorbent is used as a therapeutic agent for humanpatients by oral administration, various formulas, which are currentlyemployed in medicines used by oral administration, can be applied: forexample, a powdered or suspended powdered form, capsule, tablet andsolution. These formulas can be provided using authentic methods bymixing the endotoxin-adsorbent with various vehicles and additiveswithin a range that is acceptable with respect to pharmaceuticalguidelines.

The endotoxin-adsorbent can be administered orally at 10 mg-10 g peradult by a single or three administrations per day.

EXAMPLES

The individual examples of this invention are described in detail in thefollowing sections, but the invention is not to be considered limited tothese examples as described below.

Example 1 Epoxyacryl Resin-Polymixin B Conjugate (RPMB)

Polymixin B sulfate (3 million units, Maruko Pharmaceuticals) wasdissolved in 200 mL of 0.1M NaCl, and then the pH was adjusted to 8 byadding NaOH. Four grams of a polyacryl resin with an epoxy residue(Amberzyme, Rohm and Haas, USA) was added to the solution and stirredusing a blade propeller for 72 hours. The resin was washed with 1000 mLof distilled water on a membrane filter with a 5 μm pore size, andsuspended in 50 mL of 1M glycine solution, pH 8.0, adjusted by NaOH.After incubation overnight, the resin was washed with 2 liters ofdistilled water on a filter, and dried in a desiccator. The yield ofpolymixin B-conjugated resin (this is called as RPMB) was 3.9 g.

Example 2 Epoxyacryl Resin-Polymixin B Conjugate (RPMB-1)

Polymixin B sulfate (3 million units, Maruko Pharmaceuticals) wasdissolved in 200 mL of 0.1M NaCl, and pH adjusted to 8 by adding NaOH.Four grams of a polyacryl resin with an epoxy residue (Amberzyme, Rohmand Haas, USA) was transferred into a mortar, and ground with a pestleby adding polymixin B sulfate solution drop-wise. The Polymixin B andepoxyacryl resin were stirred for 72 hours using a magnetic stirringbar. The resin was washed with 1 liter of distilled water on a membranefilter with a 5 μm pore size, then suspended in 50 mL of 1M glycinesolution, pH 8.0, adjusted with NaOH. After incubation overnight, theresin was washed with 2 liters of distilled water on a filter and driedin a desiccator. The yield of polymixin B-conjugated resin (this iscalled as RPMB-1) was 3.2 g.

Example 3 Epoxyacryl Resin-Polymixin B Conjugate (RPBM-2)

One gram of a polyacryl resin containing an epoxy residue (Amberzyme,Rohm and Haas, USA) was ground with a mortar and pestle. The powderedresin was suspended in 20 mL of distilled water, reacted with 0.6 g ofepichlorohydrin and 0.3 mL of 50% NaOH for 2 hours. The resin was washedwith 100 mL of distilled water on a membrane filter with a 5 μm poresize, and then mixed with in 5 mL of 1M phosphate buffer, pH 10.0,containing 3 million units of polymixin B, and stirred at 40° C. for 16hours. After the reaction, the resin suspension was added by 50 mL of 1Mglycine solution, pH 8.0, adjusted with NaOH, and kept overnight. Theresin was washed with 2 liters of distilled water on a filter and driedin a desiccator. The yield of polymixin B-conjugated resin (this iscalled RPMB-2) was 0.5 g.

Example 4 Weakly Acidic Cation-Exchange Resin-Polymixin B Conjugate

Four grams of a weakly acidic cation-exchange resin with carboxylresidues (Dowex MAC-3), was suspended in 50 mL of 0.1M MOPS(3-morpholinopropanesulfonic acid) solution, pH 7.5, and then reactedwith 1 g of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(Sigma, USA), a coupling agent, at 4° C. for 2 hours with stirring. Theactivated resin was collected on a membrane filter with a 5 μm pore sizeand washed with 200 mL of distilled water. The washed activated resinwas then suspended in 50 mL of 0.1M MOPS, pH 7.5, and then mixed with 3million units of polymixin B dissolved in 10 mL of 0.1M MOPS solution,pH 7.5, and reacted at 4° C. for 16 hours with stirring. The resin wascollected on a membrane filter with a 5 μm pore size and then suspendedin 50 mL of 1M glycine solution, pH 8.0, adjusted with NaOH, and kept at4° C. overnight, washed with 2 liters of distilled water and then driedin a desiccator. The yield of polymixin-conjugated weakly acidic cationexchange resin (This preparation is called 4/300) was 3.8 g.

Example 5 Weakly Acidic Cation-Exchange Resin-Polymixin B Conjugate(1/300)

Using the same resin (Dowex MAC-3) and same procedures shown in theExample 4, except for reducing the amount of resin from 4 to 1 g, 0.9 gof polymixin B-conjugated weakly acidic cation exchange resin wasobtained. This preparation is called 1/300.

Example 6 Weakly Acidic Cation-Exchange Resin-Polymixin B Conjugate(4M/300)

Four grams of weakly acidic cation-exchange resin with carboxyl residues(Dowex MAC-3), was ground with a mortar and pestle, and suspended in 50mL of 0.1M MOPS, pH 7.5. The resin was reacted with 1 g of1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (Sigma,USA), a coupling agent, at 4° C. for 2 hours with stirring. Theactivated resin was collected on a membrane filter with a 5 μm pore sizeand washed with 200 mL of distilled water. The resin was suspended in 50mL of 0.1M MOPS, pH 7.5, and then added to 10 mL 0.1M MOPS, pH 7.5,containing 3 million units of polymixin B. Polymixin B and the resinwere reacted at 4° C. for 16 hours with stirring. The resin wascollected on a membrane filter with a 5 μm pore size and then suspendedin 50 mL of 1M glycine solution, pH 8.0, adjusted with NaOH, and keptovernight, washed with 2 liters of distilled water, and then dried in adesiccator. The yield of polymixin B-conjugated weakly acidic cationexchange resin was 3.1 g. This preparation is called 4M/300.

Example 7 Weakly Acidic Cation-Exchange Resin-Polymixin B Conjugate(1M/300)

Using the same resin and same procedures as described in Example 6,except for reducing the amount of resin from 4 to 1 g, 0.52 g ofpolymixin B-conjugated weakly acidic cation exchange resin was obtained.This preparation is called 1M/300.

Example 8 Large Scale Grinding of Resin

Dowex Mac-3 resin (diameter: 300-1200 mm), 3300 liters, was ground usinga Dalton NeaMill, NEA-48 type. The yield of powdered Mac-3 was 1400 kgand the average particle size was 30 μm, ranging from 10 to 50 μm.

Example 9 Measurement of Particle Size Distribution

The particle size distribution was measured according to “Measurement ofParticle Size Distribution. Method 1: Microscopic method” in the secondsupplement of the general test procedures, Section 65, JapanesePharmacopoeia, 13^(th) Issue. The microscope and camera used for thisexperiment was Nikon ECLIPSE E600 and Victor KY-F55B, respectively. Thecollected data was analyzed using Nano Hunter NS2K-Pro. The result ofanalysis of 1006 particles of RPME prepared in Example 1 by this methodis shown in Table 1. The 50% particle size of RPMB was 213 μm, and thecontent of small particles not more than 5 μm in a diameter was 0%.

TABLE 1 Distribution of Particles Size of RPMB* Distribution of ParticleSize Diameter 10% Diameter 160 (μm) 50% Diameter 213 (μm) 90% Diameter266 (μm) Average Diameter 197 (μm) *Total 1006 particles were analyzed.

Example 10 Endotoxin Adsorption and Elimination Capacity (1)

Endotoxin was assayed by an end point colorimetric assay method usingEndospecy-ES24S kit (Seikagaku Kogyo, Japan). Lipopolysaccharide (LPS)from E. coli O-111 (Sigma L4130) was dissolved in pyrogen-free water at5 μg/mL. One mL of this LPS solution was mixed with 50 μg and 100 μg ofRPMB-1, and 100 μg of polymixin B-unconjugated resin (control) andincubated at 37° C. with stirring. Endotoxin levels were determined inthe supernatant before, 10 and 20 minutes after adding the resins. Asshown in Table 2, LPS was specifically adsorbed by RPMB-1.

TABLE 2 Endotoxin removal activity of RPMB determined by limulus assayLPS Activity (EU/mL) Amount Remaining Resin (μg) Before 10 mm 20 mmRPMB-l 50 5632 2403 1207 RPMB-l 100 5526 1004 170 Control 100 5711 46054988 Resin

Example 11 Endotoxin Adsorption and Elimination Capacity (2)

The endotoxin-binding capacity of polymixin B conjugates was alsostudied. LPS (Sigma L4130) was dissolved in pyrogen-free water at 0.2mg/mL, and 4 mL of this solution was added to a test tube containing20.6 mg of RPMB-1, and incubated at 37° C. with stirring. Thesupernatant was collected every 30 minutes by centrifugation and the ODvalues at 210 nm was determined. The OD210 value was dropped from 1 to0.6 within the first 30 minutes of incubation and remained unchangedafterwards. By adding 20.5 mg of fresh RPM-1 into the supernatant, theOD value was slightly reduced from 0.6 to 0.4. Therefore, it was assumedthat 20.6 mg of LPS adsorbent added in the first test tube was saturatedwith LPS. Accordingly, an LPS adsorption capacity of RPMB-1 wascalculated based on the OD value changes of LPS solution. Since the LPSpreparation used in this experiment was not pure and contaminated by DNAand proteins, it was assumed that the final OD value of 0.4 reflectedthe OD value of such contaminants. Based on this assumption, it wascalculated that 1 g of RPMB is capable of binding approximately 25.9 mgof LPS (Sigma L4130) using the following formula:(0.8 mg×0.4/0.6)/0.0206=25.9 mgSince the endotoxin unit of this LPS preparation is 1.1×10⁶ EU/mg, itwas calculated that 1 g of RPMB-1 is capable of binding 29×10⁶ EU ofendotoxin.

Example 12 Effect of Particle Size on Endotoxin-Binding Capacity

Polymixin B-conjugated resins prepared in Examples 1-7 were analyzed fortheir particle size distribution by the method described in Example 9and assayed for the endotoxin-binding capacity by the method describedin Example 11 to study the relationship between particle size andLPS-binding capacity. AffiPrep poplymixin B (BioRad, USA) was used as areference.

The endotoxin-binding capacity of individual batches ofendotoxin-adsorbents prepared by conjugating with 3 million units ofpolymixin B was compared and expressed as endotoxin units (EU) per gramweight of resin as well as LPS weight per gram resin. The weight of LPSwas obtained by converting the EU values based on the EU value per mg ofLPS preparation (Sigma L4130, LPS preparation from E. coli O-111, B4, bytrichloroacetic acid extraction) used for this experiment.

In spite of using the same amount of 3 million units of polymixin B tomake conjugates as described in Example 1-7, it was apparent that theendotoxin-binding capacity of polymixin B-conjugated resin is higher ifthe 50% particle size is smaller as shown in Table 3.

TABLE 3 Relationship between particle sizes and endotoxin-bindingcapacity 50% Endotoxin- Endotoxin- Particle Binding Binding Polymixin B-Size Capacity capacity Conjugated Resins (μm) (EU/g) (LP5 mg/g*)AffiPrep 61 14 × 10⁶ 12 (mg) Polymixin B (Reference) RPMB 213 20 × 10⁶17.5 (mg) RPMB-1 32 29 × 10⁶ 26.5 (mg) RPMB-2 32 120 × 10⁶  105 (mg)4/300 400 7.2 × 10⁶  6.3 (mg) 1/300 400 7.7 × 10⁶  6.7 (mg) 4M/300 2629.5 × 10⁶   30 (mg) 1M/300 29 136.3 × 10⁶   120 (mg) *Converted to dryweight of LPS

Example 13 Evaluation of Endotoxin-Adsorbent in Autoantibody Mediated,LPS-Induced Arthritis Model

DBA/1JNCrj mice (Japan Charles River) were divided into 5 groups (G1-G5,5 mice per group). In order to increase the mucosal permeability, allmice received 40 μg of indomethasin (Sigma) and 2 mg of ovomucoid(Sigma) for 5 consecutive days from day −6 to −2 by the oral route. Onday 0, all mice received an IV injection of 0.2 mL of arthritogenicmonoclonal antibody cocktail (10 mg/mL). Endotoxin derived from E. coliO-111 (Phenol extracted LPS, Sigma) was dissolved in PBS at 7.5 mg/mL,and 0.2 mL of this solution was administered into G1-G4 mice by the oralroute for 3 consecutive days from day 0 to 2. Endotoxin-adsorbent,RPMB-1, was suspended in distilled water at 100 mg/mL, and deaerated bya vacuum pump to keep the particles in uniform suspension by preventingthe aggregation of the particles. The RPMB-1 suspension was administeredto mice at doses of 0.125 mL (G2), 0.25 mL (G3) and 0.5 mL (G4) twice aday for 4 consecutive days from day 0 to 3 after LPS administration. G1received 0.25 mL of water alone. Mice in G5, a positive control ofarthritis, received IP injection of 0.1 mL of LPS solution (0.5 mg/mL inPBS) on day 3.

All mice were observed for the development of arthritis every day fromday 0 to 14. Severity of arthritis was scored by 5 grades, 0: normalwithout any swelling, 1: clinically apparent swelling of one digit, 2:moderate redness and swelling of more than 2 digits or moderate rednessand swelling of the entire paw, 3: severe redness and swelling of theentire paws, and 4: maximum inflamed limb with involvement of multiplejoints. The sum of the arthritis score (maximum 16 per mouse) ofindividual animals was calculated. The effect of the endotoxin-adsorbentwas calculated based on the average score of 5 mice using the followingequation:Suppression of arthritis(%)=(1−Average score of test group/Average scoreof control group)×100

Since the arthritis scores reached a maximum on day 7, the effect of theendotoxin-adsorbent was calculated using the scores on day 7. Thesuppression of arthritis by RPMB prepared in EXAMPLE 2 at 25 mg, 50 mgand 100 mg per mouse by oral administration is shown in Table 3. None ofthe five mice which received 100 mg of RPMB-1 developed arthritis,whereas 2 out of 5 mice which received 50 mg of RPMB-1 developed mildarthritis (average score: 2), and 4 out of 5 mice which received 25 mgof RPMB-1 developed moderate arthritis (average score: 9) (Table 3,experiment 1), indicating a dose response effectiveness of RPMB-1.

Similarly, RPMB-2, 1M/300 and 4M/300, which have higher bindingcapacities of LPS then RPMB-1, were also tested for their effect onarthritis at a dose of 10 mg per mouse. All three preparations wereequally effective and suppressed the development of arthritis almostcompletely (Table 3, experiment 2)

TABLE 3 Suppression of arthritis by polymixin B conjugated resinsPolymixin B- Incidence Severity of Experiment Conjugated Dose ofArthritis No Resin (mg/mouse) Arthritis (score) 1 Control 50 5/5  12 ±1.6 Resin RPMB-1 25 4/5 6.4 ± 1.8 RPMB-1 50 2/5   2 ± 2.8 RPMB-1 100 0/50 ± 0 2 Control 10 5/5 12.6 ± 1.1  Resin RPMB-2 10 0/5 0 ± 0 4M/300 101/5 0.8 ± 1.8 1M/300 10 0/5 0 ± 0

Example 14 Tablet

The tablets were prepared by mixing 15 g of 1M/300, which was preparedin EXAMPLE 7, 2.5 g of lactose, 2.4 g of corn starch, and 0.1 g ofmagnesium stearate. These four components were mixed well and compressedby a single punch tableting machine to make tablets containing 200 mg of1M/300 per a tablet.

Example 15 Capsule

RPMB-2 powder shown in EXAMPLE 3 was dispensed into hard capsules at 150mg per a capsule.

1. A method of treating rheumatoid arthritis in a patient havingrheumatoid arthritis comprising the step of orally administering to thepatient a pharmacologically effective amount of an endotoxin-adsorbentcomprising a Lipid A-binding molecule having an endotoxin-bindingactivity and particles carrying the Lipid A-binding molecule, whereinthe Lipid A-binding molecule is polymixin B, and the particles carryingthe Lipid A-binding molecule comprise a synthetic polymer having anepoxy residue, and wherein the particles comprise indigestible particlesin which no more than 1% of the particles are 5 μm or less in diameterand more than 90% of the particles are 50 μm or less in diameter, basedon volume-based distribution analysis.
 2. The method of claim 1, whereinthe particles carrying the endotoxin-binding active group comprises apolyacryl resin having an epoxy residue.
 3. The method of claim 1,wherein the particles carrying the endotoxin-binding active groupcomprises a polystyrene resin having epoxy residue.
 4. The method ofclaim 1, wherein the synthetic polymer is selected from the groupconsisting of polyacrylate, polystyrene, polypropylene, polyamide andpolyvinyl.